Polymeric materials that elicit controlled cell responses, and have good mechanical, optical and/or biodegradation properties, are disclosed for use in biomedical applications. Processing methods by which such polymers can be localized at a biomaterial surface are also disclosed.
Polymers currently in use for biomedical applications generally tend to be hydrophobic. As defined herein, hydrophobic refers to a material that repels water, i.e., exhibits a static contact angle with water greater than 60 degrees at 20.degree. C., and has a water permeability P less than 3.times.10.sup.-10 cm.sup.3 (STP) cm/(cm.sup.2 s Pa). This can give rise to uncontrolled interactions between cells and adsorbed proteins at the surface of the material, which can result in a chronic inflammatory response that can lead to failure of implants and even promote tumorigenecity (Warson, The Applications of Synthetic Resin Emulsions, Benn, London (1972)). Metal or ceramic materials used in implant applications similarly can elicit undesirable cell responses.
For tissue engineering applications, it is essential that the polymeric material used to form a biodegradable scaffold for cells promote cell adhesion, migration, growth and differentiation while providing adequate structural support. Though commonly used synthetic scaffold materials such as poly(lactide), poly(glycolide), etc., and copolymers thereof, have suitable mechanical, processing and biodegradation properties, their hydrophobic nature leads to protein adsorption and denaturing on the material surface which elicits uncontrolled cell response.
The ideal surface for many biomaterials applications would resist protein adsorption while providing cells with specific chemical signals to guide adhesion, survival, growth, migration and differentiation. As used herein, the term "biomaterial" refers to a nonviable material used in a medical device intended to interact with biological systems. Polymer surfaces modified with poly(ethylene oxide) have been studied in recent years for the reduction of protein adsorption at the surface of biomaterials (Paine et al. Macromolecules, 23:3104 (1990)). The objective of these surface modification schemes is the elimination of nonspecific interactions of cells with implant materials. One way in which specific chemical signals can be relayed to cells at a surface is through tethered ligands for cell surface receptors (Barret, Brit. Polym. J. 5:259 (1973)). Delivery of signals in this manner has advantages over the addition of soluble factors, as the signal is presented in a very localized manner at a controlled dose without diffusive loss (Kuhl and Griffith, Nature Medicine, 2:1002 (1996)). In addition, tethered ligands may provide more constant stimulation to cells by avoiding the down-regulation present when soluble ligands are internalized by cells. Control over spatial distribution of ligands on surfaces may also be key to guiding cell behavior. Thus systems which will allow spatial control of local ligand density, or the creation of clusters of ligands on a surface, in addition to providing control over the average surface density of ligands, are highly desirable (Kornberg et al, Proc. Natl. Acad. Sci. USA, 88:8392 (1991)).
Integrins, dimeric adhesion receptors including one of approximately ten known alpha chains paired with one of approximately six known beta chains, mediate a wide range of interactions between cells and extracellular matrix (ECM) and control cell behaviors as diverse as migration, growth, and differentiation, providing a permissive environment for the action of growth factors. For many integrins, the specificity of integrin binding to matrix proteins has been mapped to small, discrete peptide domains and new sites continue to be elucidated (Rouslahti, Ann. Rev. Cell. Dev. Biol., 12: 697 (1996); Hynes, Cell, 48:549 (1987)). The prototypical example of such specificity is the RGD site first identified in fibronectin and subsequently identified in other matrix proteins. The RGD peptide enables complete replacement of adhesive function of fibronectin for cells expressing certain integrins.
Much data supports the idea that both occupancy and clustering of integrins are required to elicit full cellular responses mediated by integrins (Clark and Brugge, Science, 268:233 (1995)). For example, fill EGFR activation of MAP kinase requires integrin clustering and occupancy (Miyamoto et al, J. Cell Biol., 135:1633 (1996)). Thus, the spatial presentation of ligand in the environment, i.e., whether ligands are spaced closely enough to afford clustering of ligand-bound integrins, may influence cellular behaviors governed by integrins. Indeed, spacing of synthetic RGD ligand covalently linked to the substrate has been shown to have an influence on cell adhesion and spreading (Massia and Hubbell, J. Cell Biol., 114:1089 (1991)). At the same time, the surface concentration of an adhesion ligand such as fibronectin has been shown to have a substantial influence on integrin-mediated behaviors such as migration (DiMilla et al, J. Cell Biol., 122:729 (1993)). A recent study using self-assembled monolayers patterned in one micron adhesive/nonadhesive domains demonstrated the role of cell spreading and receptor occupancy on cell survival (Chen et al, Science, 276:1425 (1997). The length scale in that study was approximately that of a focal adhesion complex (or larger), but it is likely that clustering over much smaller length scales (3-10 integrins) is also physiologically relevant. Indeed, data suggests strongly that RGD clustering on the less than 100 nm length scale has profound effects on the integrin-mediated behavior of migration. Since both the concentration and spatial distribution of ligand influence cell response, it is desirable to have a means to vary these two parameters independently, and over a broad range of length scales (nanometers to micrometers), in order to guide cell response.
Integrins can initiate intracellular signaling cascades that overlap with those of growth factors such as epidermal growth factor (EGF). Cross-communication between adhesion and growth factor receptors may occur by direct physical association within the focal adhesions. Both types of receptors are concentrated in these structures (Miyamoto et al, J. Cell Biol., 135:1633 (1996); Plopper et al, Mol. Biol. Cell, 6:1349 (1995)), and both receptors can stimulate some of the same down-stream effect on molecules such as MAP kinase. Close proximity of adhesion and growth factor receptors in the focal adhesion complex provides for a free flow of both positive and negative regulatory signals between the two. A number of signaling molecules have been proposed as forming this linkage; one intracellular mechanism of transmodulation is via protein kinase C (PKC)-mediated attenuation of the epidermal growth factor receptor (EGFR). It is also likely that PKC activity secondary to phospholipase C.sub..gamma. or phospholipase D activation by EGFR alters integrin-based substratum connections (Welsh et al, J. Cell Biol., 114:533 (1991); Ando et al, J. Cell. Physiol., 156:487 (1993)). It is thus desirable to have a method by which two or more types of signaling ligands, such as adhesion peptides and growth factors, can be simultaneously located at the surface of a biomaterial in controlled quanitity and spatial distribution.
To date, few if any model systems are able to meet both protein resistance and cell signaling surface requirements, while approaches using clinically-applicable materials have focused on hydrogels (Hern and Hubbell, J. Biomed. Mater. Res., 39:266 (1998)), which have limited physical strength and are not suitable for many applications. Other approaches for modifying the surfaces of hydrophobic polymeric materials or other biomaterials to achieve a more desirable surface composition for biomedical applications include adsorption of block copolymers, chemical grafting of polymers to the surface, and plasma deposition of an overlying film. Each of these methods suffers various disadvantages. For example, adsorbed block copolymers can be rearranged actively by cells, grafted polymers are difficult to apply at high density on a surface, and plasma deposition results in a gel-like surface structure poorly suited for controlled cell signaling. None of these methods provides a means for modifying the surface of complex three-dimensional structures such as fibrous or sponge-like tissue scaffolds, or for creating clustered ligand distributions of variable concentration and spacing on biomaterial surfaces.
It would be advantageous to provide polymer materials and processing methods that overcome the disadvantages of other biomaterials surface modification approaches. It is therefore an object of the present invention to provide polymer materials that elicit controlled cell-surface interactions by inhibiting protein adsorption, and, where appropriate, presenting controlled concentrations and spatial distributions of cell-signaling ligands on biomaterial surfaces. It is further an object of the present invention to provide processing methods by which such polymers can be placed at a biomaterial surface. It is further the object of the present invention to provide polymeric materials which can be used to create discrete nanometer- to micrometer-sized domains on a biomaterial surface that present two or more different types of ligands for regulating cellular response.